Subdivision of flowstreams into two or more paths is a frequent requirement in modern chemical instrumentation. The apparatus used to create the separate flowstreams is generally referred to as a flow splitter. Frequently flow splitters are used to present some representative portion of the output of a flow system to an electronic detector, typically to measure the progress of some chemical processing step [e.g. separation, extraction or reaction progress].
Analytical liquid chromatography is used for separation of complex mixtures of solutes dissolved in a liquid solvent. A high pressure pumping system is used to force a liquid mobile phase through a separation column packed with very small, high surface area silica spheres that have been chemically modified to interact at differing degrees with different types of solutes. A strong interaction between the modified silica surface and a solute will cause the solute to be retained at the surface for a longer period than a weak interaction. Thus, for a mixture of solutes, those having a weaker interaction with the silica surface will emerge from the separation column in a shorter period of time than those with strong interactions.
The liquid mobile phase also plays a significant role in the time a particular solute spends on the separation column. Weak solvents are those that are unable to remove solutes from the separation surface. Strong solvents are those that readily redissolve solutes that are even strongly adsorbed on the surface. For a mixture with a wide range of solutes that interact differently, a technique called gradient elution is used. In this case, the original mobile phase starts at a composition that makes it a very weak solvent. Gradually, the composition is changed by increasing the relative concentration of strong solvent. The process continues until a maximum composition is reached or all solutes have been eluted from the column.
The result is that a complex mixture of solutes introduced to a separation column emerge as individual solutes at different times and in different flow segments of the mobile phase based on the strength of their interaction with the modified silica surface and the mobile phase. With the solutes separated into different flow segments of the mobile phase flowstream, the flow can be directed through an electronic detector which can sense presence of specific types of solutes based on the solutes' physical properties [e.g. molecular mass, UV-visible light absorption, refractive index, electrochemical reduction or oxidation potential, etc.]. Such detectors generate an electronic signal that in analytical chromatography is simply used to quantitate the amount of solute present, or qualify the presence or absence of a particular component in the mixture. Because of the greatly improved performance of using high pressure pumps through packed columns of very small particles, the modern technique has become known high performance liquid chromatography or HPLC.
Preparative high performance liquid chromatography extends the art of analytical liquid chromatography by adding a collection step to the instrument system. In this case, the electronic signals generated by one or more detectors are used to trigger the collection of specific segments of the mobile phase flowstream that contain the desired solutes. By isolating only these specific desired flow segments, a purification is performed which competes economically with other purification methods such as recrystallization. In order to be efficient, preparative HPLC systems must be scaled up to significantly higher flow rates, solute concentrations and column sizes in order to process significant amounts of material.
The solute concentrations used in preparative HPLC are disproportionately high compared to analytical HPLC. For example, a typical analytical HPLC separation may apply 5 microliters (μl) of sample mixture to the analytical separation column in a mobile phase flowstream of 1 mL/min. The solute concentrations of the mixture are typically in the range of 100 μg/mL, so a total of 500 nanograms are applied into the system. Further, it is common to split only a fraction ranging from 5% to 35% of the analytical mobile phase directly to the split detector. In contrast, users frequently inject 100 mg samples in one to two milliliter volumes into preparative HPLC systems scaled to only 20 times the column size and flow rate of the analytical system. Thus while the applied sample mass increases up to 200,000 fold and sample volume increases by 200 fold, the flow rates only increase 20 fold over the analytical separation. This means that local concentrations are as much as 1000 times higher in the preparative experiment. This presents a serious problem since the vast majority of electronic detectors for chromatography are designed for use with analytical concentrations of solutes.
The commercial solution to this problem of very high solute concentrations in preparative HPLC mobile phases is to use a flow splitting device to partition off a very small fraction of the main chromatographic flow stream, apply a diluent and deliver the diluted sample stream to a detector such as a mass spectrometer. Most manufacturers of preparative equipment supply a flow splitter for this reason. In practice, such splitting schemes can typically provide split ratios as high as 10,000:1 and dilute samples up to 100 fold.
The application of conventional flow splitters has been largely unsuccessful in preparative supercritical fluid chromatography (SFC) applications. In SFC, a gas such as carbon dioxide (CO2) is compressed to liquid-like densities and used as the major component of the chromatographic mobile phase. Carbon dioxide has a critical temperature of 31 degrees Celcius and critical pressure of 73.8 bar. When raised above the critical temperature and pressure, CO2 is no longer considered a liquid, but rather a supercritical fluid that has liquid-like densities and solvating power. The solvating power can be greatly enhanced by addition of organic liquids in solution with the CO2. Use of varying compositions of CO2 and methanol, for example, give a range of solvent strength from approximately hexane for pure CO2 to nearly that of pure methanol in a 50% solution of Methanol/CO2.
The advantages of preparative SFC are numerous and include faster separation, higher loading capability and lower energy requirements for the desolvation of collected fractions. Faster separation is achieved principally due to the significantly lowered viscosity of the SFC mobile phase. Unlike HPLC where water is the principle component with a viscosity of ˜1 centipoise (cp) under standard conditions, supercritical CO2 has a viscosity of ˜0.05 cp at standard conditions of 100 bar and 40 deg C. The result from a chromatography standpoint is much lower pressure across separation columns even at high flow rates and much greater diffusion rates into and out of the columns separating medium. Since diffusion is the major rate limiting property of the chromatographic process, the entire separation process speeds up as much as 20 fold compared to similar HPLC separations.
Preparative SFC systems require a high pressure backpressure regulator to maintain the CO2 at liquid-like densities that keep it soluble with organic liquids and solutes. Typically the backpressure setting ranges from 100 bar to 300 bar.
The mobile phase of SFC is subject to phase change, including up to a 500-fold volume expansion and significant temperature drop as the pressure goes from 100 bar to atmospheric pressure as it passes through traditional restrictor-based flow splitters prior to entry into the detector. This localized change of phase, flow, temperature and viscosity within the splitter restrictor makes the split behavior unpredictable, especially over the range of compositions and concentrations found during gradient chromatography. Further, the low diameter tubing required is subject to plugging, especially when the evaporating CO2 carries with it some of the organic solvent solvating the solutes in the mobile phase. This evaporative solvent loss coupled with the intensly cold temperatures can easily result in precipitation of solutes from the remaining organic solvent in the tubing.
Construction of a useful flow splitter must take into account the physical properties of the mobile phase that is to be sampled. Such properties may include, for example, the flow rate, viscosity, phase (e.g. gas, liquid, supercritical), pressure, or composition variation (change in concentration of solvent, change in solutes eluted) over time. Each of these elements may affect the performance of the flow splitter. In addition, the flow and concentration requirements of the target detector or detectors must be taken into account.
What is needed is a flow-splitting device that can reliably control the split ratio from a primary flowstream from moderate to very high split ratios while diluting sample concentrations to levels appropriate to the target detectors. At the same time such a device must overcome the problems associated with fixed splitters including the effects of pressure and viscosity variations and phase change in the region where the actual flow splitting occurs. Finally, the device must be able to operate in a continuous manner at maximum pressures higher than 100 bar, for example up to about 300 bar, and eventually deliver the split flow to an atmospheric pressure device. The device should be suitable for both HPLC and SFC type mobile phases.